Non-IgA fc binding forms of the Group B streptococcal beta antigens

ABSTRACT

The invention relates to a mutant Cβ protein comprising the amino acid sequence A-X 1  X 2  X 3  X 4  X 5  X 6  X 7  X  8  X 9  X 10  X 11  X 12 -B, wherein A comprises amino acids 1-164 of the sequence shown in FIG.  1  (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG.  1  (SEQ ID NO: 2), and X 1 -X 12  are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG.  1  (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X 1  through X 12 , inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein. The invention also relates to a polynucleotide molecule encoding a mutant Cβ protein, as well as vectors comprising such polynucleotide molecules, and host cells transformed therewith. The invention also relates to a conjugate comprising the mutant Cβ protein covalently conjugated to a capsular polysaccharide. The invention also relates to a vaccine comprising at least one mutant Cβ protein of the invention and a pharmaceutically acceptable carrier. The invention also relates to a method of inducing an immune response in an animal, comprising administering the vaccine of the invention to an animal in a therapeutically effective amount.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention concerns the construction of a protein having a reduced or eliminated ability to bind human IgA, but that retains the immunological properties useful for formulating a conjugate vaccine against Group B streptococci.

[0003] 2. Related Art

[0004] Streptococci are a large and varied set of gram-positive bacteria which have been ordered into several groups based on the antigenicity and structure of their cell wall polysaccharide (Lancefield, R. C., J. Exp. Med. 57: 571-595 (1933); Lancefield, R. C., Proc. Soc. Exp. Biol. and Med. 38: 473-478 (1938)). Two of these groups have been associated with serious human infections. Those that have been classified into Group A streptococci are the bacteria that people are most familiar and are the organisms which cause “strep throat.” Organisms of Group A streptococci also are associated with the more serious infections of rheumatic fever, streptococcal impetigo, and sepsis.

[0005] Group B streptococci were not known as a human pathogen in standard medical textbooks until the early 1970's. Since that time, studies have shown that Group B streptococci are an important perinatal pathogen in both the United States as well as the developing countries (Smith, A. L. and J. Haas, Infections of the Central Nervous System, Raven Press, Ltd., New York. (1991) p. 313-333). Systemic Group B streptococcal infections during the first two months of life affect approximately three out of every 1000 births (Dillon, H. C., Jr., et al., J. Pediat. 110: 31-36 (1987)), resulting in 11,000 cases annually in the United States. These infections cause symptoms of congenital pneumonia, sepsis, and meningitis. A substantial number of these infants die or have permanent neurological sequelae. Furthermore, these Group B streptococcal infections may be implicated in the high pregnancy-related morbidity which occurs in nearly 50,000 women annually. Others who are at risk from Group B streptococcal infections include those who either congenitally, chemotherapeutically, or by other means, have an altered immune response.

[0006] Group B streptococci can be further classified into several different types based on the bacteria's capsular polysaccharide. The most pathogenically important of these different types are streptococci having types Ia, Ib, II, or III capsular polysaccharides. Group B streptococci of these four types represent over 90% of all reported cases. The structure of each of these various polysaccharide types has been elucidated and characterized (Jennings, H. J., et al., Biochemistry 22: 1258-1263 (1983); Jennings, H. J., et al., Can. J. Biochem. 58: 112-120 (1980); Jennings, H. J., et al., Proc. Nat. Acad. Sci. USA. 77: 2931-2935 (1980); Jennings, H. J., et al., J. Biol. Chem. 258: 1793-1798 (1983); Wessels, M. R., et al., J. Biol. Chem. 262: 8262-8267 (1987)). As is found with many other human bacterial pathogens, it has been ascertained that the capsular polysaccharides of Group B streptococci, when used as vaccines, provide very effective, efficacious protection against infections with these bacteria. This was first noted by Lancefield (Lancefield, R. C., et al., J. Exp. Med. 142: 165-179 (1975)) and more recently in the numerous studies of Kasper and coworkers (Baker, C. J., et al., N. Engl. J. Med. 319: 1180-1185 (1988); Baltimore, R. S., et al., J. Infect. Dis. 140: 81-86 (1979); Kasper, D. L., et al., J. Exp. Med. 149: 327-339 (1979); Madoff, L. C., et al., J. Clin. Invest. 94: 286-292 (1994); Marques, M. B., et al., Infect. Immun. 62: 1593-1599 (1994); Wessels, M. R., et al., J. Clin. Invest. 86: 1428-1433 (1990); Wessels, M. R., et al., Infect. Immun. 61: 4760-4766 (1993); Wyle, S. A., et al., J. Infect. Dis. 126: 514-522 (1972)). However, much like many other capsular polysaccharide vaccines (Anderson, P., et al., J. Clin. Invest. 51: 39-44 (1972); Gold, R., et al., J. Clin. Invest. 56: 1536-1547 (1975); Gold, R., et al., J. Infect. Dis. 136S: S31-S35 (1977); Gold, R. M., et al., J. Infect. Dis. 138: 731-735 (1978); Mäkelä, P. R. H., et al., J. Infect. Dis. 136: S43-50 (1977); Peltola, A., et al., Pediatrics 60: 730-737 (1977); Peltola, H., et al. N. Engl. J. Med. 297: 686-691 (1977)), vaccines formulated from pure type Ia, Ib, II, and III capsular carbohydrates are relatively poor immunogens and have very little efficacy in children under the age of 18 months (Baker, C. J. and D. L. Kasper. Rev. Inf. Dis. 7: 458-467 (1985); Baker, C. J., et al., N. Engl. J. Med. 319: 1180-1185 (1988); Baker, C. J., et al., New Engl. J. Med. 322: 1857-1860 (1990)). These pure polysaccharides are classified as T cell independent antigens because they induce a similar immunological response in animals devoid of T lymphocytes (Howard, J. G., et al., Cell. Immunol. 2: 614-626 (1971)). It is thought that these polysaccharides do not evoke a secondary booster response because they do not interact with T cells, and therefore fail to provoke a subsequent “helper response” via the secretion of various cytokines. For this reason, each consecutive administration of the polysaccharide as a vaccine results in the release of a constant amount of antibodies, while a T cell dependent antigen would elicit an ever increasing concentration of antibodies each time it was administered.

[0007] Goebel and Avery found in 1931 that by covalently linking a pure polysaccharide to a protein that they could evoke an immune response to the polysaccharide which could not be accomplished using the polysaccharide alone (Avery, O. T. and W. F. Goebel, J. Exp. Med. 54: 437-447 (1931); Goebel, W. F. and O. T. Avery, J. Exp. Med. 54: 431-436 (1931)). These observations initiated and formed the basis of the current conjugate vaccine technology. Numerous studies have followed and show that when polysaccharides are coupled to proteins prior to their administration as vaccines, the immune response to the polysaccharides changes from a T independent response to a T dependent response (see (Dick, W. E., Jr. and M. Beurret, Glycoconjugates of bacterial carbohydrate antigens In: Contributions to Microbiology and Immunology. Cruse et al., eds., (1989) p.48-114; Jennings, H. J. and R. K. Sood, Neoglycoconjugates: Preparation and Applications. Y. C. Lee and R. T. Lee, eds., Academic Press, New York. (1994) p. 325-371; Robbins, J. B. and R. Schneerson, J. Infect. Dis. 161: 821-832 (1990)) for reviews). Currently, most of these polysaccharide-protein conjugate vaccines are formulated with well known proteins such as tetanus toxoid and diphtheria toxoid or mutants thereof. These proteins were originally used because they were already licensed for human use and were well characterized. However, as more and more polysaccharides were coupled to these proteins and used as vaccines, interference between the various vaccines which used the same protein became apparent. For example, if several different polysaccharides were linked to tetanus toxoid and given sequentially, the immune response to the first administered polysaccharide conjugate would be much larger than the last. If, however, each of the polysaccharides were coupled to a different protein and administered sequentially, the immune response to each of the polysaccharides would be the same. Carrier suppression is the term used to describe this observed phenomenon. One approach to overcome this problem is to match the protein and polysaccharide so that they are derived from the same organism.

[0008] Among the various antigens used to classify and subgroup Group B streptococci, one was a protein known as the Ibc antigen. This protein antigen was first described by Wilkinson and Eagon in 1971 (Wilkinson, H. W. and R. G. Eagon, Infect. Immun. 4: 596-604 (1971)) and was known to be made up of two distinct proteins designated as alpha and beta. Later, the Ibc antigen was shown to be effective when used as a vaccine antigen in a mouse model of infection by Lancefield and co-workers (Lancefield, R. C., et al., J. Exp. Med. 142: 165-179 (1975)). The isolation, purification and functional characterization of the beta antigen (Cβ) protein of Group B streptococci was accomplished by Russell-Jones, et al. (Russell-Jones, G. J. and E. C. Gotschlich, J. Exp. Med. 160: 1476-1484 (1984); Russell-Jones, G. J., et al., J. Exp. Med. 160: 1467-1475 (1984)) [see U.S. Pat. No. 4,757,134]. They could demonstrate that one of the properties of the Cβ protein was to bind specifically to human IgA immunoglobulin. The binding site on the IgA molecule was localized to the Fc portion of the heavy chain of this immunoglobulin. They further showed that the Cβ protein consisted of a single polypeptide having an estimated molecular weight of 130,000 daltons. The gene responsible for the expression of the Cβ protein was cloned (Cleat, P. H. and K. N. Timmis, Infect. Immun. 55: 1151-1155 (1987)) and sequenced (Jerlström, P. G., et al., Molec. Microbiol. 5: 843-849 (1991)) by a group led by Timmis. His later study demonstrated that the IgA binding activity could be assigned to a 746 bp DNA fragment of the gene defined by a leading BglII restriction endonuclease cleavage site and ending with a HpaI restriction endonuclease cleavage site.

[0009] As stated previously, the 1975 Lancefield study showed that the Ibc antigen was an effective vaccine antigen in a mouse model of Group B streptococcal infection (Lancefield, R. C., et al., J. Exp. Med. 142: 165-179 (1975)). It was not clear at the time whether the alpha or beta protein component of the Ibc antigen was responsible for this protection. Madoff et al., began to shed light on this question and demonstrated that the purified Cβ protein used as a vaccine could protect infant mice from experimental infection with Group B streptococci expressing this protein (Madoff, L. C., et al., Infect. Immun. 60: 4989-4994 (1992)). Madoff et al., then went on to show that when they coupled a Type III streptococcal capsular polysaccharide to the Cβ protein, producing a conjugate vaccine, this vaccine would protect infant mice against infection with either a Type III Group B streptococci (expressing no Cβ) or a Type Ib Group B streptococci (expressing Cβ but lacking a Type III capsular polysaccharide) (Madoff, L. C., et al., J. Clin. Invest. 94: 286-292 (1994)). Thus, such a Cβ protein conjugate vaccine served several functions: the polysaccharide elicited protective antibodies to the polysaccharide capsule and the Cβ protein evoked protective antibodies to the protein as well as modified the immune response to the polysaccharide from a T independent response to a T dependent response.

[0010] This polysaccharide-Cβ protein conjugate strategy works well in mice. But clearly, the goal is to protect humans against Group B streptococcal infections. The only caveat with using the same strategy in humans is that the Cβ protein binds human IgA immunoglobulins non-specifically (Cβ does not bind mouse IgA). This human IgA binding activity of Cβ could diminish the efficacy of a polysaccharide-Cβ protein conjugate vaccine for humans, as antigens bound to IgA can be cleared from the system so rapidly that an antigen-specific antibody response is not produced. Furthermore, potentially protective epitopes on the Cβ protein could be hidden when the human IgA binds to the Cβ molecule. Thus, it would be advantageous to obtain a mutant Cβ protein which lacks the IgA binding capacity but retains as much of the native structure as possible.

[0011] With this goal in mind, several groups have attempted to determine the IgA binding region of the Cβ protein. Jerlström et al. (Molec. Microbiol. 5: 843-849 (1991)) used experiments wherein subfragments of the Cβ protein were expressed as fusion proteins to identify two regions of the Cβ protein capable of binding IgA. These experiments localized the IgA binding domains to a 747 bp BglII-HpaI fragment and a 1461 bp HpaI-HindIII fragment of the Cβ protein. Furthermore, International Patent Application No. PCT/US/06111 describes the isolation of a Cβ protein bearing a deletion of a region that binds IgA.

SUMMARY OF THE INVENTION

[0012] The invention relates to a mutant Cβ protein, wherein the IgA binding by the Cβ protein is reduced or eliminated, while the antigenicity of the protein when administered either alone or as part of a polysaccharide-protein conjugate is substantially retained.

[0013] In particular, the invention relates to a mutant Cβ protein comprising the amino acid sequence A-X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂-B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X₁-X₁₂ are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X₁ through X₁₂, inclusive, is other than the wild type amino acid.

[0014] The invention also relates to a polynucleotide molecule encoding a mutant Cβ protein, as well as vectors comprising such polynucleotide molecules, and host cells transformed therewith.

[0015] The invention also relates to a conjugate comprising the mutant Cβ protein covalently conjugated to a capsular polysaccharide.

[0016] The invention also relates to a vaccine comprising the mutant Cβ protein of the invention and a pharmaceutically acceptable carrier.

[0017] The invention also relates to a method of inducing an immune response in an animal, comprising administering the vaccine of the invention to an animal in an effective amount.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows the DNA sequence and deduced amino acid sequence of wild type Cβ1 (Jerlström, P. G., et al., Molec. Microbiol. 5: 843-849 (1991)). The BglII and PstI sites shown in FIGS. 2, 3 and 4 are identified.

[0019]FIG. 2 is a map of the region of the Cβ gene which encodes the IgA binding site of the Cβ protein; 2 amino acid substitutions are indicated, generating mutant dgb2 (see Table 1).

[0020]FIG. 3 is a map of the region of the Cβ gene which encodes the IgA binding site of the Cβ protein; 2 amino acid substitutions are indicated, generating mutant nv34qp (see Table 1).

[0021]FIG. 4 is a map of the region of the Cβ gene which encodes the IgA binding site of the Cβ protein; 6 amino acids have been deleted from this region in the mutant protein, generating mutant dgb1 (see Table 1).

[0022]FIG. 5 is a graph showing the competitive inhibition of ELISA reactivity by Cβ proteins.

[0023]FIGS. 6A, 6B and 6C show the complete DNA sequence of the gene encoding Cβ mutant dgb2 (see Table 1), as well as the deduced amino acid sequence of this mutant. The mutations are underlined.

[0024]FIGS. 7A, 7B and 7C show the complete DNA sequence of the gene encoding Cβ mutant nv34qp (see Table 1), as well as the deduced amino acid sequence of this mutant. The mutations are underlined.

[0025]FIGS. 8A, 8B and 8C show the complete DNA sequence of the gene encoding Cβ mutant dgb1 (see Table 1), as well as the deduced amino acid sequence of this mutant.

[0026]FIGS. 9A, 9B and 9C show the complete DNA sequence of the gene encoding Cβ mutant pnv231 (see Table 1), as well as the deduced amino acid sequence of this mutant. The mutations are underlined.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The invention relates to a mutant Cβ protein of the group B streptococcal (GBS) beta antigen, wherein IgA binding by the Cβ protein is reduced or eliminated and wherein at least a majority of the antigenicity of the protein is retained.

[0028] It has been discovered that mutation of a region of the Cβ protein located between about amino acid residues 163 and 176 of the wildtype Cβ sequence shown in FIG. 1 (SEQ ID NO: 2) results in a Cβ protein which has reduced or eliminated IgA binding properties, but which retains enough of its tertiary structure to maintain the majority of its antigenicity (see Examples 4 and 5).

[0029] As the region of the Cβ polypeptide has been found which is responsible for IgA binding, and as it has been demonstrated in the Examples below that amino acid substitutions or deletions in this region reduce or eliminate IgA binding while maintaining antigenicity of the protein, those of ordinary skill in the art will understand how to alter the amino acid sequence of the Cβ polypeptide so as to achieve the objects of the invention. Appropriate amino acid substitutions which eliminate IgA binding will include replacement of one or more residues with an amino acid having different properties. For example, a strongly hydrophilic amino acid can be replaced with a strongly hydrophobic amino acid. Amino acids which can be grouped together include the aliphatic amino acids Ala, Val, Leu and Ile; the hydroxyl residues Ser and Thr, the acidic residues Asp and Glu, the amide residues Asn and Gln, the basic residues Lys and Arg and the aromatic residues Phe and Tyr. Thus, those of ordinary skill in the art will understand how to determine suitable amino acid substitutions or deletions in the region between about residues 163 and 176 in the Cβ protein in order to reduce or eliminate IgA binding.

[0030] Further guidance concerning which amino acid changes are likely to have a significant deleterious effect on a function can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247: 1306-1310 (1990).

[0031] Thus, in particular, the invention relates to a mutant group B streptococcal (GBS) beta antigen, Cβ, comprising the amino acid sequence A-X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂-B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X₁-X₁₂ are each selected independently from the group consisting of Ala, Arg, Asp, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X₁ through X₁₂, inclusive, is other than the wild type amino acid. In a particularly preferred mutant Cβ protein, amino acids X₇ and X₁₂ are Ala (SEQ ID NO: 3). In another preferred mutant, amino acids X₄ and X₁₁ are Pro (SEQ ID NO: 4). In another preferred mutant, amino acid X₇ is Thr and amino acid X₁₂ is Leu (SEQ ID NO: 5). In a more preferred mutant, amino acids X₅, X₇, X₈, X₁₀, X₁₁ and X₁₂ are each replaced with a bond (SEQ ID NO: 6).

[0032] As the Cβ protein is, in its wild type state, membrane bound, it is possible to improve purification of the above-mentioned Cβ mutants by eliminating the hydrophobic residues of the transmembrane domain of the Cβ protein (the transmembrane domain corresponds to residues 1095-1127 of the sequence shown in FIG. 1 (SEQ ID NO: 2)). This can be accomplished by substitution of non-hydrophobic residues for the hydrophobic residues (residues 1108-1116 of the sequence shown in FIG. 1 (SEQ ID NO: 2)) or by deletion of the hydrophobic residues. While purification of membrane-bound Cβ requires the use of detergent, a mutant Cβ which lacks the hydrophobic membrane spanning region can be purified without using detergent. Thus, the invention also relates to a mutant Cβ wherein the nine hydrophobic residues making up the transmembrane domain are deleted or replaced by non-hydrophobic amino acids.

[0033] It has been discovered that the IgA-binding ability of Cβ may require dimerization of Cβ. Thus, even where the IgA-binding region of Cβ is not mutated as described above, mutation of the region of Cβ which is believed to be required for dimerization can result in a form of Cβ that cannot bind IgA. Deletion of a portion of Cβ from residue 729 to the C-terminus of the sequence shown in FIG. 1 (SEQ ID NO: 2) eliminates dimerization of Cβ. The results of experiments supporting this finding may be found in Table 1. Several fragments of Cβ were inserted into each of two different vectors. Where sequences shown in the table are preceded or followed by an outward facing bracket, this indicates that the Cβ sequence does not extend further on that end of the fragment., i.e. that the nucleotide sequence inserted into the vector encodes only those amino acids shown, and no more of the Cβ sequence. Where sequences shown in the table are preceded or followed by ellipses, this indicates that the remainder of the Cβ sequence at that end of the fragment is also included in the vector. Nucleotide sequences encoding the peptides shown in the upper part of the table were inserted into either the vector pTOPE or the vector pET17b. Both of these vectors allow expression of inserted fragments from the T7 promoter, and both produce fusion proteins containing a fragment of the φ10 capsid protein N terminal to the amino acid sequence encoded by the insert. However, while pET17b encodes only 8 amino acids of the φ10 protein, pTOPE encodes a 288 amino acid fragment of the φ10 protein.

[0034] As shown in Table 1, certain fragments of Cβ produced from pET17b exhibit reduced IgA-binding, while the same fragment produced by pTOPE is capable of binding IgA. The fragments tested lack the region of Cβ predicted to be involved in dimerization, but do not contain any mutations in the putative IgA binding domain (note that the Cβ fragments inserted into vector pET24b, shown at the bottom of Table 1, contain the putative dimerization region but nonetheless exhibit reduced IgA binding due to mutations in the IgA binding domain, as described above). It is postulated that these Cβ fragments bind DNA when produced from pTOPE because the 288 amino acid fragment of the φ10 protein allows dimerization of the Cβ fragment. This may be due to the fact that the φ10 capsid protein normally forms oligomers; the region responsible for oligomerization may thus allow dimerization of the inserted Cβ fragments, and thus IgA-binding. Thus, the invention also relates to a mutant Cβ protein having a mutation in the dimerization domain of Cβ, wherein the mutant Cβ protein is incapable of binding IgA. Of course, in the interest of producing a non-IgA binding Cβ protein retaining as much of the antigenicity of the wild type Cβ protein as possible, dimerization of Cβ should not be interrupted.

[0035] It has also been discovered that production of Cβ protein from E. coli can be problematic because the protein is cleaved at a specific region, presumably by an E. coli signal peptidase. This cleavage results in a truncated protein, which obviously is not ideal for a vaccine, as it lacks many antigenic epitopes of the wildtype Cβ protein. The cleavage site has been predicted by sequence analysis and by matrix assisted laser desorption initiated time of flight (MALDI-TOF) mass spectrometry (von Heijne, Nucleic Acids Res. 14: 4683-4690 (1986)). The cleavage site is between amino acid residues 538 and 539 (after alanine and before glutamine) of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). The signal peptidase recognition site is located within a 20 amino acid stretch located between residues 521 and 541 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). Therefore, by deleting this region, the Cβ protein or a non-IgA binding mutant thereof can successfully be produced in E. coli. Furthermore, as signal peptidases have very strict sequence specificity, alteration of the signal peptidase recognition sequence, including even a single, conservative amino acid substitution in this region, may eliminate cleavage of Cβ by E. coli. The recognition sequence required for cleavage by this signal peptidase is believed to be GluLeuIleLysSerAlaGlnGlnGlu (SEQ ID NO: 1), corresponding to amino acid residues 533-541 of the sequence shown in FIG. 1 (SEQ ID NO: 2). Alteration of either the serine or the alanine residue of this sequence by either deletion or non-conservative substitution is expected to eliminate cleavage by the signal peptidase. Of course, ideally, the mutagenesis of Cβ will be kept to a minimum so as to retain the tertiary structure of the wildtype antigen for the purposes of eliciting an immunogenic response.

[0036] Thus, the invention also relates to a mutant Cβ protein of the group B streptococcal (GBS) beta antigen, wherein IgA binding by the Cβ protein is reduced or eliminated by any of the mutations described above, and wherein at least one of amino acid residues 521-541 of the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) is either (a) deleted or (b) altered, so that the protein is not cleaved in this region when Cβ is produced in E. coli. In a preferred embodiment, at least one of amino acid residues 533-541 of the sequence shown in FIG. 1 (SEQ ID NO: 2) is either (a) deleted or (b) altered. In a more preferred embodiment, at least one of amino acid residues 537 and 538 is either (a) deleted or (b) altered. Of course, one of ordinary skill will be able to determine other suitable amino acid substitutions by routine experimentation, and by reference to the article by von Heijne (Nucleic Acids Res. 14: 4683-4690 (1986)).

[0037] The invention also relates to polynucleotide molecules encoding the mutant proteins of the invention, vectors comprising those polynucleotide molecules, and host cells transformed therewith.

[0038] The invention also relates to the expression of novel mutant Cβ polypeptides, wherein IgA binding by the Cβ protein is reduced or eliminated, in a cellular host.

[0039] Prokaryotic hosts that may be used for cloning and expressing the polypeptides of the invention are well known in the art. Vectors which replicate in such host cells are also well known.

[0040] Preferred prokaryotic hosts include, but are not limited to, bacteria of the genus Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, Xanthomonas, etc. Two such prokaryotic hosts are E. coli DH10B and DH5αF′IQ (available from LTI, Gaithersburg, Md.). The most preferred host for cloning and expressing the polypeptides of the invention is E. coli BL21 (Novagen, Wis.), which is lysogenic for DE3 phage.

[0041] The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of the polypeptides of the invention by recombinant techniques.

[0042] Host cells can be genetically engineered to incorporate nucleic acid molecules and express polypeptides of the present invention. For instance, recombinant constructs may be introduced into host cells using well known techniques of infection, transduction, transfection, and transformation. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.

[0043] Thus, for instance, the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. The vector construct may be introduced into host cells by the aforementioned techniques. Generally, a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. Electroporation also may be used to introduce polynucleotides into a host. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells. A wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length in Sambrook et al. cited above, which is illustrative of the many laboratory manuals that detail these techniques.

[0044] In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsulated virus by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.

[0045] Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate transacting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

[0046] In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art (see U.S. Pat. No. 5,464,758).

[0047] The engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.

[0048] A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain or propagate, polynucleotides, or to express a polypeptide, in a host may be used for expression in this regard.

[0049] The appropriate DNA molecule may be inserted into the vector by any of a variety of well-known and routine techniques. In general, a DNA molecule for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase. Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill in the art. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited above.

[0050] The DNA molecule inserted in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the art in the manner illustrated by the discussion and the examples herein.

[0051] In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0052] In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.

[0053] Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.

[0054] The vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells. Hosts for of a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention.

[0055] More particularly, the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above. The constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted. The sequence may be inserted in a forward or reverse orientation. In certain preferred embodiments in this regard, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.

[0056] As the invention concerns the construction of a protein having a reduced or eliminated ability to bind human IgA, the invention thus relates to using in vitro mutagenesis methods to generate the mutant Cβ proteins of the invention. A number of in vitro mutagenesis methods are well known to those of skill in the art; several are provided here as examples.

[0057] One such method introduces deletions or insertions into a polynucleotide molecule inserted into a plasmid by either partially or completely digesting the plasmid with an appropriate restriction enzyme, and then ligating the ends to again generate a plasmid. Very short deletions can be made by first cutting a plasmid at a restriction site, and then subjecting the linear DNA to controlled nuclease digestion to remove small groups of bases at each end. Precise insertions may also be made by ligating double stranded oligonucleotide linkers to a plasmid cut at a single restriction site.

[0058] Chemical methods can also be used to introduce mutations to a single stranded polynucleotide molecule. For example, single base pair changes at cytosine residues can be created using chemicals such as bisulfite, which deaminates cytosine to uracil, thus converting GC base pairs to AT base pairs.

[0059] Preferably, oligonucleotide directed mutagenesis will be used so that all possible classes of base pair changes at any determined site along a DNA molecule can be made. In general, this technique involves annealing a oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence of interest. The mismatched oligonucleotide is then extended by DNA polymerase, generating a double stranded DNA molecule which contains the desired change in sequence on one strand. The changes in sequence can of course result in the deletion, substitution, or insertion of an amino acid if the change is made in the coding region of a gene. The double stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant polypeptide can thus be produced. The above-described oligonucleotide directed mutagenesis can of course be carried out via PCR. An example of such a system is the Ex-Site™ PCR site-directed mutagenesis technique (Stratagene, Calif.) used in Example 4.

[0060] Using the Ex-Site™ PCR site-directed mutagenesis technique, several different oligonucleotides were made to induce different changes in the DNA sequence in the region of interest. In one particular example, overlapping primers were obtained, wherein both primers contained the sequence required to change lysine to alanine at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID NO: 2) (see FIG. 2 and Table 1). The forward primer, designated Cβ 613, had the sequence (SEQ ID NO: 6) 5′-GTT GAA GCA ATG GCA GAG CAA GCG GGA ATC ACA AAT GAA G-3′ and the reverse primer, designated Cβ 642R had the sequence (SEQ ID NO: 7) 5′-GAT TCC CGC TTG CTC TGC CAT TGC TTC AAC TTG ACT TTT TTG-3′ (the substitutions are noted in BOLD). These oligonucleotides were combined with pNV222 template, which consists of the Cβ gene inserted into the pSP76 vector. PCR was performed, and the products were ligated and introduced into E. coli strain DH5α, thus generating clones containing the mutant Cβ gene.

[0061] The following vectors, which are commercially available, may be used in the practice of the invention. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia; pUC18, pUC19 and pPROEX-1, available from LTI, and pTOPE, pET17b, and pET24a (Novagen, Madison, Wis.). These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.

[0062] Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase (“CAT”) transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the CAT gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.

[0063] Among known bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lacI and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.

[0064] Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art.

[0065] The present invention also relates to host cells containing the constructs discussed above. The host cell can be a prokaryotic cell, such as a bacterial cell.

[0066] Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

[0067] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, where the selected promoter is inducible, it is induced by appropriate means (e.g., temperature shift or exposure to chemical inducer) and cells are cultured for an additional period.

[0068] Cells typically are then harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0069] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well know to those skilled in the art.

[0070] The invention also relates to a vaccine comprising a mutant Cβ protein, wherein IgA binding by the Cβ protein is reduced or eliminated as described herein, together with a pharmaceutically acceptable carrier. In a preferred embodiment, the protein is conjugated to a polysaccharide.

[0071] The conjugates of the invention may be formed by reacting the reducing end groups of the polysaccharide to primary amino groups (that is, lysine residues) of the Cβ protein by reductive amination. The polysaccharide may be conjugated to any or all of the primary amino groups of the protein. The reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both. Preferably, the Cβ protein is conjugated to the polysaccharide by the method of Jennings et al., U.S. Pat. No. 4,356,170, which involves controlled oxidation of the polysaccharide with periodate followed by reductive amination with the Cβ protein of the invention.

[0072] In a preferred embodiment, the polysaccharide is one of the Group B streptococcal capsular polysaccharides selected from types Ia, II, III and V. See Baker, C. J. and D. L. Kasper. Rev. Inf. Dis. 7: 458-467 (1985); Baker, C. J., et al., N. Engl. J. Med. 319: 1180-1185 (1988); Baker, C. J., et al., New Engl. J. Med. 322: 1857-1860 (1990). The vaccine may also be a combination vaccine comprising one or more of the Cβ protein-polysaccharide conjugates selected from the group consisting of Cβ conjugated to Group B capsular polysaccharide type Ia (Cβ-Ia); Cβ conjugated to Group B capsular polysaccharide type II (Cβ-II); Cβ conjugated to Group B capsular polysaccharide type III (Cβ-III); and Cβ conjugated to Group B capsular polysaccharide type V (Cβ-V). Most preferably, the vaccine is a combination vaccine comprising Cβ-Ia, Cβ-II, Cβ-III and Cβ-V. Such a combination vaccine will elicit antibodies to Group B streptoccoci of Types Ia, II, III, V, and Ib (as Type Ib Group B streptococci also express Cβ). Furthermore, the immune response to the polysaccharides of the combination vaccine will be a T dependent response.

[0073] The vaccine of the present invention comprises one or more of the Cβ protein vaccines or conjugate vaccines in amounts effective depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the vaccine of the present invention can also be administered by an intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. With respect to each conjugate, suitable amounts are expected to fall within the range of 2 micrograms of the protein per kg body weight to 100 micrograms per kg body weight. In a preferred embodiment, the vaccine comprises about 2 μg of the Cβ protein or an equivalent amount of the protein-polysaccharide conjugate. In another preferred embodiment, the vaccine comprises about 5 μg of the Cβ protein or an equivalent amount of the protein-polysaccharide conjugate.

[0074] The vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the non-IgA Fc binding group B streptococcal Cβ protein or conjugate vaccine have suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.

[0075] The vaccines of the present invention may further comprise adjuvants which enhance production of Cβ-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Pat. No. 4,258,029), the dipeptide known as MDP, saponin (see U.S. Pat. No. 5,057,540), aluminum hydroxide, and lymphatic cytokine.

[0076] Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administration to a human. The Cβ protein vaccine or a conjugate vaccine thereof may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Pat. No. 4,235,877.

[0077] In another preferred embodiment, the present invention relates to a method of inducing an immune response in an animal comprising administering to the animal the vaccine of the invention, produced according to methods described, in an amount effective to induce an immune response.

[0078] Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES Example 1 Cloning and Expression of the Gene Encoding Cβ

[0079] To locate the IgA binding site on the Cβ protein, two oligonucleotides were synthesized. The first oligonucleotide, oligo 1, corresponds to the 5′ end of the mature protein, and has the sequence (SEQ ID NO: 8) 5′-AAGGATCCAAGTGAGCTTGTAAAGGACGAT-3′, which includes a BamHI site. The second oligonucleotide falls just short of the 3′ end of the gene, and has the sequence (SEQ ID NO: 9) 5′-AAAACTCGAGTTTCTTCCGTTGTTGATGTA-3′, and includes a XhoI site. The oligonucleotide for the 3′ end of the gene was chosen to eliminate the LPXTG motif found in most gram positive cell wall proteins. This sequence motif has been shown to be involved in the processing of these cell wall proteins and is the part of these proteins which eventually becomes covalently bound to peptidoglycan (Navarre, W. W. and O. Schneewind, Molec. Microbiol. 14: 115-121 (1994); Schneewind, O., et al., Science 268: 103-106 (1995)). Using chromosomal DNA from Strain A909 Group B streptococci containing the gene for the Cβ protein as a template, and standard PCR procedures, a product of approximately 3.2 kb was produced as observed when electrophoresed on a 1% agarose gel. The PCR product containing the Cβ protein gene was cleaved with the endonuclease restriction enzymes BamHI and XhoI. This BamHI-XhoI DNA fragment contained the sequence for the entire Cβ protein except for the last 33 amino acids at the carboxyl terminus, including the putative IgA binding site. The DNA fragment was then ligated into the appropriately restricted T7 expression plasmid pET17b (Novagen Inc., Madison Wis.) using a standard T4 ligase procedure. The plasmid was then transformed into the E. coli strain BL21(DE3) using the manufacturer's suggested protocols (Novagen Inc.). E. coli cells containing the plasmid were selected on LB plates containing 50 μg/ml carbenicillin. These plates were incubated overnight at 37° C. The transformant colonies were carefully lifted onto nitrocellulose filters saturated with IPTG. After 30 min, the bacteria were lysed by placing the filters into a chloroform vapor chamber for 15 min at room temperature.

[0080] After the filters were removed from the chamber, they were placed, colony-side up, onto a Whatman 3MM filter which had been previously saturated with 20 mM Tris-HCl, pH 7.9, 6 M urea, and 0.5 M NaCl. After 15 min, the filters were washed three times in PBS and incubated for 1 hr with purified human IgA in PBS-Tween. The filters were then rewashed in the PBS-Tween and developed by standard procedures (Blake, M. S., et al., Analyt. Biochem. 136: 175-17 (1984)) using a goat antihuman IgA-alkaline phosphatase conjugate (Cappel Research Products, West Chester, Pa.). Several colonies demonstrating high IgA binding activity were selected and grown overnight in 1 ml LB broth containing carbenicillin at 30° C. These cultures were then diluted 1 to 100 with fresh LB-carbenicillin broth and incubated at 30° C. for an addition 6 hr. Expression was then induced by the addition of IPTG and the culture continued for an addition 2 hr at 30° C. The cells were collected by centrifugation, resuspended in water and subjected to several freeze-thaw cycles. The cells were once again collected by centrifugation and the supernatants saved for examination of their IgA binding activity.

Example 2 Identification of the IgA Binding Domain of Cβ

[0081] Once certain a stable plasmid producing a recombinant Cβ protein had been achieved and that the expressed protein bound human IgA, a strategy similar to that of the Novatope System (Novagen, Inc.) was utilized to locate the IgA binding region of Cβ. This procedure was performed according to the manufacturer's instructions. Briefly, the purified plasmid containing the Cβ gene was randomly digested with DNase I and electrophoresed in a 2% low melting point agarose gel. Fragments of the DNA corresponding to sizes between 100 to 300 base pairs were excised from the gel, purified, and resuspended in TE buffer. A single dA was added to the fragments using the recommended reaction mixture and the fragments ligated into the pETOPET vector which contained single dT ends. After the standard ligation procedure, the plasmids were transformed into competent NovaBlue (DE3) cells (Novagen, Inc.) and plated on LB plates containing 50 μg/ml carbenicillin. These plates were incubated overnight at 37° C. The transformant colonies were tested for IgA binding activity as described in Example 1. Several clones were selected on the bases of their binding to the IgA. The bacteria from each of these clones were inoculated separately onto fresh LB plates and retested for their IgA binding ability as before. Plasmid preparations were made from each by standard means and sequenced.

[0082] The nucleotide sequences of the cloned Cβ protein gene fragments were determined by the dideoxy method using denatured double-stranded plasmid DNA template as described (Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (1993)). Sequenase II kits (United States Biochemical Corp., Cleveland, Ohio) were used in accordance with the manufacturer's instructions. The smallest fragment of DNA obtained that included part of the Cβ gene is shown in FIG. 1. The translation of this sequence corresponds to amino acid 101 to 230 of the mature Cβ protein shown in FIG. 1 (SEQ ID NO: 2). Attempts to further shorten this DNA fragment failed to give any IgA binding activity.

Example 3 ELISA Inhibition Assays: Peptide Binding Studies

[0083] Several synthetic peptides were made corresponding to the amino acid sequence contained within this region of the Cβ protein. Peptides were synthesized using NMP t-butoxycarbonyl chemistry on an ABI 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and were deprotected. Peptides from a sample of the resin were removed from the resin by treatment with HF in the presence of anisole (0° C./1 h). Preparative purification of these peptides were performed using a C18 column (2.14 ID×30 cm) (Dynamax-Rainin, Woburn, Mass.). The peptides were quantitated by PTC amino acid analysis using Waters Picotag system (Waters, Milford, Mass.). The synthesized peptides eluted from the C18 column as a major peak consisting of usually 75-85% of the total elution profile. The amino acid composition of the purified peptides were in good agreement with the sequence which was used to synthesize the peptides. These peptides were used in ELISA inhibition assays to block the binding of human IgA to the purified Cβ protein as follows. Microtiter plates (Nunc-Immuno Plate IIF, Vangard International, Neptune, N.J.) were sensitized by adding 0.1 ml per well of purified Cβ at a concentration of 2.0 μg/ml in 0.1 M Carbonate buffer, pH 9.6 with 0.02% azide. The plates were incubated overnight at room temperature. The plates were washed five times with 0.9% NaCl, 0.05% Brij 35, 10 mM sodium acetate pH 7.0, 0.02% azide. A purified human IgA myeloma protein was purchased from Cappel Laboratories, was diluted in PBS with 0.5% Brij 35 and added to the plate and incubated for 1 hr at room temperature. The plates were again washed as before and the secondary antibody, alkaline phosphatase conjugated goat anti-human IgA (Tago Inc., Burlingame, Calif.), was diluted in PBS-Brij, added to the plates and incubated for 1 h at room temperature. The plates were washed as before and p-nitrophenyl phosphate (Sigma phosphatase substrate 104) (1 mg/ml) in 0.1 M diethanolamine, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.02% azide, pH 9.8 added. The plates were incubated at 37° C. for 1 h and the absorbance at 405 nm determined using an Elida-5 microtiter plate reader (Physica, New York, N.Y.). Control wells lacked either the primary and/or secondary antibody. This was done to obtain a titer of the human IgA myeloma protein which would give a half-maximal reading in the ELISA assay. This titer would be used in the inhibition ELISA. The microtiter plate were sensitized and washed as before. Purified synthetic peptides were added and diluted in PBS-Brig. The dilution of the human IgA myeloma protein which gave the half maximal reading was then added. The mixture was then incubated for 1 hr at room temperature. The plates were rewashed and the conjugated second antibody added as stated. The plates were then processed and read as described. The percentage of inhibition would be calculated as follows:

[0084] 1−(ELISA value with the peptide added)/(ELISA value without the peptide added).

[0085] The peptide which inhibited in this ELISA assay contained the sequence Asn-His-Gln-Lys-Ser-Gln-Val-Glu-Lys-Met-Ala-Glu-Gln-Lys-Gly (SEQ ID NO: 10). This suggested that at least part of the IgA binding domain of the Cβ was comprised within the region of the protein containing this sequence.

Example 4 Oligonucleotide Directed Mutagenesis of the Gene Encoding Cβ

[0086] In order to confirm the importance of this region in the Cβ protein for IgA binding activity and to begin to generate the mutant proteins that in the end would be used in the vaccine formulation, a modification of the Ex-Site™ PCR site-directed mutagenesis protocol was employed as developed by Stratagene (Stratagene, Calif.). The template used was a plasmid called pNV222 which consisted of the Cβ gene inserted into the pSP76 vector (Promega, Madison, Wis.). DNA oligonucleotides were synthesized on an Applied Biosystems model 292 DNA Synthesizer (Foster City, Calif.). The oligonucleotides were manually cleaved from the column by treatment with 1.5 ml of ammonium hydroxide for 2 hours with gentle mixing every 15 minutes. They were deprotected at 55° C. for 16-18 hours. After deprotection they were dried down and used directly or purified using oligonucleotide purification columns (Applied Biosystems, Foster City Calif.). Several different oligonucleotides were made to induce different changes in the DNA sequence in the region of interest. An example of which is the following. The primers, in this particular example, were overlapping primers, both containing the sequence required to change lysine to alanine at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID NO: 2). The forward primer, designated Cβ 613, had the sequence (SEQ ID NO: 6) 5′-GTT GAA GCA ATG GCA GAG CAA GCG GGA ATC ACA AAT GAA G-3′ and the reverse primer, designated Cβ 642R had the sequence (SEQ ID NO: 7) 5′-GAT TCC CGC TTG CTC TGC CAT TGC TTC AAC TTG ACT TTT TTG-3′ (the substitutions are noted in BOLD). The reaction conditions were as follows: 10 ng pNV222 template, 15 pmol. of each primer, 1 mM of each dNTP, 1X VENT Polymerase Buffer (20 mM Tris-HCl, pH 7.5; 10 mM KCl; 10 mM (NH₄)₂ SO₄; 2 mM MgSO₄ 0.1% (v/v) Triton® X-100; 0.1 mg/ml bovine serum albumin (BSA)), 10 units of Vent Polymerase, and H₂O to 100 μl. The reactions were prepared with PCR Gem 10 wax beads as per the Hot Start Protocol (Perkin Elmer, Foster City, Calif.). The reactions were run in a Perkin Elmer Thermocycler (Perkin Elmer, Foster City, Calif.) under the following conditions: 1 cycle of 94° C. for 5 minutes; 10 cycles of 94° C. for 30 seconds, 37° C. for 2 minutes, 72° C. for 10 minutes; 30 cycles of 94° C. for 30 seconds, 55° C. for 2 minutes, 72° C. for 10 minutes; and 1 cycle of 72° C. for 12 minutes. The reaction was treated with 10 units of DpnI at 37° C. for 30 minutes to destroy the template DNA, followed by a 60 minute treatment at 72° C. with PfuI polymerase to fill in any remaining overhangs. The reaction was diluted 1:4.6 in 1 X Vent Buffer plus 0.38 mM dATP. The diluted reaction was ligated for 24 hours at 25° C. and transformed into competent DH5α cells (Gibco/BRL, Gaithersburg, Md.). Selected colonies were grown in 3 ml of LB plus kanamycin (50 mg/ml) at 37° C. for 16-18 hours. DNA was prepared using QIAspin™ columns (Qiagen, Chatsworth, Calif.). The clones were analyzed for insert size on 0.8% agarose gels and then sequenced. Selected clones were then grown in 100 ml LB plus kanamycin (50 mg/ml) at 37° C. for 16-18 hours. DNA was prepared using the Qiagen-tip 100 (Qiagen, Chatsworth, Calif.). They were then digested with NdeI and PstI and run on 0.8% agarose gels to separate the mutated region. The 2300 bp fragment was isolated and purified from the gel using the Gene-Clean Spin Kit™ (Bio 101, Vista, Calif.). A clone named pNV34 which consisted of the expression vector pET 24a (Novagen) and the native Cβ gene, was also digested with NdeI and PstI and run on a 0.8% agarose gel. The large band (6300 bp) containing the pET vector and the remainder of the Cβ gene was isolated and purified from the gel using the Gene-Clean Spin Kit™ (Bio 101). These two fragments were ligated at 4° C. for 24 hours and transformed into competant BL21 (DE3) cells. Selected colonies were grown in 3 ml of LB plus kanamycin (50 mg/ml) at 37° C. for 16-18 hours. DNA was prepared using QIAspin™ columns (Qiagen) and the clones were analyzed for insert size on 0.8% agarose gels.

[0087] Also constructed were clones encoding mutant Cβ proteins wherein two glutaminyl residues are replaced by prolinyl residues (FIG. 3), and wherein a deletion in the Cβ gene had occurred resulting in a 6 amino acid deletion in the region of interest (FIG. 4).

[0088] Clones expressing a Cβ protein which lacked or had reduced IgA binding activity but still reacted with the anti-βag antiserum were selected (see Example 5) and grown in 100 ml LB plus kanamycin (50 mg/ml) at 37° C. for 16-18 hours. Plasmid DNA from these clones was prepared using Qiagen tip 100 (Qiagen) and the mutated Cβ gene entirely sequenced.

Example 5 Western Blot and ELISA Analysis of IgA Binding by Cβ Mutants

[0089] The proteins encoded by the mutated genes were expressed and subjected to SDS-PAGE and western blot analysis in order to determine if mutations in the gene encoding the Cβ protein reduced or eliminated IgA binding, while retaining Cβ antigenicity. Two western blots were made for each sample and reacted with either the purified human IgA myeloma protein or hyperimmune rabbit anti-βag protein antiserum. The clone expressing a Cβ protein wherein lysine is changed to alanine at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID NO: 2) demonstrated almost no IgA binding activity, but the ability of the protein to react with anti-Cβ antiserum remained high. IgA binding activity was also substantially eliminated in the clone expressing a Cβ protein wherein two glutaminyl residues are replaced by prolinyl residues (FIG. 3) and in the clone encoding a Cβ protein having a six amino acid deletion (FIG. 4), while reactivity with the anti-Cβ antiserum was maintained for both. The data for the clone having a six amino acid deletion suggested that the residues responsible for the IgA binding activity of the Cβ protein were located within this region of the protein, and that other possible mutations within this area would effect the IgA binding activity.

[0090] A competitive inhibition ELISA was used to more precisely determine the amount of antigenic and/or structure change the sequence modifications had on the Cβ protein. Microtiter plates (Nunc-Immuno Plate IIF, Vangard International, Neptune, N.J.) were sensitized by adding 0.1 ml per well of purified Cβ at a concentration of 2.0 μg/ml in 0.1 M carbonate buffer, pH 9.6 with 0.02% azide. The plates were incubated overnight at room temperature. The plates were washed five times with 0.9% NaCl, 0.05% Brij 35, 10 mM sodium acetate pH 7.0, 0.02% azide. Hyperimmune rabbit antiserum to the Cβ protein was diluted in PBS with 0.5% Brij 35 and added to the plate and incubated for 1 hr at room temperature. The plates were again washed as before and the secondary antibody, alkaline phosphatase conjugated goat anti-rabbit IgG (Tago Inc., Burlingame, Calif.), was diluted in PBS-Brij, added to the plates and incubated for 1 h at room temperature. The plates were washed as before and p-nitrophenyl phosphate (Sigma Phosphatase Substrate 104) (1 mg/ml) in 0.1 M diethanolamine, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.02% azide, pH 9.8, was added. The plates were incubated at 37° C. for 1 h and the absorbance at 405 nm determined using an Elida-5 microtiter plate reader (Physica, New York, N.Y.). Control wells lacked either the primary and/or secondary antibody. This was done to obtain a titer of the rabbit anti-Cβ protein which would give a half-maximal reading in the ELISA assay. This titer would be used in the inhibition ELISA. The microtiter plate were sensitized and washed as before. Purified Cβ protein or mutations of the Cβ protein were added and diluted in PBS-Brig. The dilution of the rabbit anti-Cβ protein which gave the half maximal reading was then added. The mixture was then incubated for 1 hr at room temperature. The plates were rewashed and the conjugated second antibody added as stated. The plates were then processed and read as described. The percentage of inhibition would be calculated as follows:

[0091] 1−(ELISA value with the protein added)/(ELISA value without the proteins added).

[0092]FIG. 4 shows the results of one of these inhibition ELISA assays. In this assay the inhibition of the wildtype Cβ protein from streptococci is compared with the recombinant Cβ protein and the glutaminyl to prolinyl mutants, both expressed in E. coli. As can be seen from the figure, this assay is sensitive enough to detect the absence of the membrane spanning region in the recombinants of the Cβ proteins. However, when the recombinant Cβ protein containing the wildtype sequence is compared to the substitution mutant lacking IgA binding activity, the antigenic differences are minimal. This would suggest that such substitution mutants maintain most of the antigenic character of the Cβ protein but lack the unwanted the IgA binding activity.

[0093] Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following Claims. All patents and publications cited herein are incorporated by reference herein in their entirety. % wild- Name Sequence Vector type IgAbs+ ]LLHIKQHEEVEKDKKAKQQKTLKQSDTKVDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQA pTOPE 100 DKKEDAEVKVREELGKLFSSTKAGLDQEIQ[ dgb6 ]DSDALLELENQFNETNRLLHIKQHEEVEKDKKAKQQKTLKQSDTKVDLSNIDKELNHQKSQVEKMAEQKGITNEDK 17b DSMLKKIEDIRKQAQQAKKEDAVEVKVREELGKLFSSTKAGLDQEIQEHVKKETSSEENTQKVDEHYANSL[ dgb6p ]DSDALLELENQFNETNRLLHIKQHEEVEKDKKAKQQKTLKQSDTKVDLSNIDKELNHQKSQVEKMAEQKGITNEDK pTOPE DSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQEIQEHVKKETSSEENQKVDEHYANSL[ dgb7 ]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 17b 0 IQEHVKKETSSEENTQKVDEHYANSLQNLAQKSLE[ dgb7p ]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKREELGKLFSSTKAGLDQEI pTOPE 100 QEHVKKETSSEENTQKVDEHYANSLQNLAQKSLE[ dgb8 ]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQDKKEDAEVKVREELGKLFSSTKAGLDEIQ 17b 0 EHVKKETSSEENTQKVDEHYANLQNLAQKSLEELDKATTNE[ dgb8p ]VDLSNIDKELHNQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE pTOPE 100 IQEHVKKETSSEENTQKVDEHYANSLQNLAQKSLEELDKATTNE[ dgp10 ]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKV[ 17b 10 dgb12 ...VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKV[ 17b 20 dgb11 ...VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 17b 20 IQFHVKKETSSEENTQKVDEHYANSL[ nv34qp ...VDLSNIDKELNHQKSPVEKMAEPKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 24a 10 IQ... dgb2 ...VDLSNIDKELNHQKSQVEAMAEQAGITNEDKSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 24a 60 IQ... dgb1 ...VDLNIDKELNHQKSQE(...Δ...)AGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQEIQ... 24a 0 pnv231 ...VDLSNIDKELNHQKSQVETMAEQLGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 24a IQ... 

What is claimed is:
 1. A polynucleotide molecule comprising a nucleotide sequence that encodes a mutant Cβ protein comprising the amino acid sequence A-X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂-B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X₁-X₁₂ are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X₁ through X₁₂, inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein.
 2. The polynucleotide molecule of claim 1, wherein X₁, X₅, X₇, X₈, X₁₀, X₁₁, and X₁₂ are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), with the proviso that at least one of X₁, X₅, X₇, X₈, X₁₀, X₁₁, and X₁₂ is other than the wild type amino acid.
 3. The polynucleotide molecule of claim 1, wherein X₁ and X₁₁ are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.
 4. The polynucleotide molecule of claim 1, wherein X₁ and X₁₁ are Pro.
 5. The polynucleotide molecule of claim 1, wherein X₇ and X₁₂ are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.
 6. The polynucleotide molecule of claim 1, wherein X₇ and X₁₂ are Ala.
 7. The polynucleotide molecule of claim 1, wherein X₅, X₇, X₈, X₁₀, X₁₁ and X₁₂ are each a bond.
 8. A mutant Cβ protein comprising the amino acid sequence A-X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂-B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X₁-X₁₂ are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X₁ through X₁₂, inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein.
 9. The protein of claim 8, wherein X₁, X₅, X₇, X₈, X₁₀, X₁₁, and X₁₂ are each selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), with the proviso that at least one of X₁, X₅, X₇, X₈, X₁₀, X₁₁, and X₁₂ is other than the wild type amino acid.
 10. The protein of claim 8, wherein X₁ and X₁₁ are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.
 11. The protein of claim 8, wherein X₁ and X₁₁ are Pro.
 12. The protein of claim 8, wherein X₇ and X₁₂ are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.
 13. The protein of claim 8, wherein X₇ and X₁₂ are Ala.
 14. The protein of claim 8, wherein X₅, X₇, X₈, X₁₀, X₁₁ and X₁₂ are each a bond.
 15. The mutant Cβ protein of claim 8, wherein the hydrophobic amino acid residues 1108-1116 are replaced by non-hydrophobic amino acids.
 16. The mutant Cβ protein of claim 8, wherein at least one of amino acid residues 521-541, inclusive, of Cβ is either (a) deleted or (b)) altered, so that the protein is not cleaved in this region when produced in E. coli.
 17. The mutant Cβ protein of claim 16, wherein at least one of amino acid residues 533-541, inclusive, of Cβ is either (a) deleted or (b) altered.
 18. The mutant Cβ protein of claim 16, wherein at least one of amino acid residues 537-538 of Cβ is either (a) deleted or (b) altered.
 19. A polysaccharide-protein conjugate comprising the mutant Cβ protein of claim 8 and a streptococcal capsular polysaccharide.
 20. A vector comprising the polynucleotide molecule of claim
 1. 21. A host cell transformed with the vector of claim
 20. 22. A vaccine comprising at least one mutant Cβ protein of claim 8, together with a pharmaceutically acceptable carrier.
 23. The vaccine of claim 22, wherein said mutant Cβ protein is conjugated to a polysaccharide.
 24. The vaccine of claim 23, wherein said polysaccharide to which said mutant Cβ protein is conjugated is selected from the group consisting of Group B streptococcal capsular polysaccharide types Ia, II, III and V.
 25. A combination vaccine comprising at least two Cβ protein-polysaccharide conjugates selected from the group consisting of Cβ-Ia, Cβ-II, Cβ-III and Cβ-V, together with a pharmaceutically acceptable carrier, wherein the Cβ portion of each conjugate is the mutant Cβ of claim
 8. 26. The vaccine of claim 25, said vaccine comprising Cβ-Ia, Cβ-II, Cβ-III and Cβ-V, together with a pharmaceutically acceptable carrier.
 27. A method of inducing an immune response in a mammal, comprising administering a vaccine comprising at least one mutant Cβ protein comprising the amino acid sequence A-X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂-B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X₁-X₁₂ are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X₁ through X₁₂, inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein, together with a pharmaceutically acceptable carrier, in an amount sufficient to induce an immune response in a mammal.
 28. The method of claim 27, wherein said mutant Cβ protein is conjugated to a streptococcal capsular polysaccharide.
 29. The method of claim 28, wherein said mammal is a human. 